Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are both neurodegenerative diseases, characterized by gradual progressing symptoms, that inevitably lead to death. Both are connected to mutations in several potential genes. C9orf72 is thought to be one of the key genes involved in development of ALS/FTD. Mutation occurs in the hexanucleotide (GGGGCC) repeats region inside the first intron. Normally there are about 30 repeats, but mutation can raise that number to more than thousand. One of the main hypotheses explaining the pathological effect of extended hexanucleotide repeats is accumulation of toxic proteins with dipeptide repeats (DPR). There are five proteins DPR: poly(GA), poly(GR), poly(PA), poly(PR), poly(GP), which are produced by translation that is independent of the start codon AUG.
We prepared five cell lines that would stably express proteins DPR with 125 repeats. We used restriction to cut fragments of DNA that code for proteins DPR from previously prepared plasmid vectors and cloned them in expression plasmid vector pcDNA5 FRT TO mScarlet-I Myc stop. We wanted proteins DPR to cotranslate with fluorescent marker mScarlet, but not all proteins were in the same reading frame as the marker. For that reason we moved the reading frame of the expression vector for one and for two nucleotides. We transfected Flp-in HEK293T cell line with these constructs. Flp-in system allows us integration of DNA in the genome via Flp recombinase-mediated DNA recombination at the FRT sites, located on expression vector and cell genome. After transfection and cell selection we prepared selected colonies for microscopy and confirmed successful expression of proteins DPR with fluorescent microscopy. We also added DAPI so we could observe DPR localization in cells.
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